Autophagy is a pathway of degrading cytoplasmic materials ubiquitously observed in eucaryocytes. Autophagy is classified into three types based on its mechanisms; i.e., macroautophagy, microautophagy, and chaperone-mediated autophagy. In any pathway, cytoplasmic materials are ultimately translocated into lysosomes (vacuoles in the case of yeast or plants) and degraded by degrading enzymes existing therein. Examples of the cytoplasmic materials to be degraded include not only protein molecules but also organelles such as mitochondria and endoplasmic reticulum. Since autophagy is induced by nutrient starvation, supply of nutrient components to cells via recycling of the degraded cytoplasmic materials had been considered to be a main role thereof. However, autophagy has recently been found to be associated with various vital phenomena, such as quality control of proteins or organelles, bacterial infection, antigen presentation, cell death or apoptosis, and canceration. Since autophagy is associated with degradation and elimination of abnormal proteins that accumulate and aggregate in cells, it is suggested that autophagy is associated with neurodegenerative diseases such as Huntington's disease and Alzheimer's disease, which are considered to develop due to cell death or apoptosis caused by accumulation of abnormal proteins (Deretic, V. and Klionsky, D. J., Scientific American, Vol. 298, pp. 74-81, 2008). Under such circumstances, there are needs for developing a simple and accurate method for measuring autophagy with the aim of elucidating mechanisms of the vital phenomena or developing methods for treating diseases associated with such mechanisms.
In the past, autophagy had been measured by: observing cells under electron microscope; measuring an activity of an enzyme designed to be activated specifically upon degradation of a radioisotope-labeled protein or upon autophagy; or other techniques. However, such techniques required skills and times due to insufficient specificity for autophagy and complicated procedures.
In recent years, techniques for applying fluorescent proteins represented by GFP have been advanced, and techniques for labeling autophagy-associated proteins with a fluorescent protein and measuring their phamacokinetics by fluorescence methods such as microscopic imaging and flow cytometry has become generalized. Such techniques enabled simple measurement of autophagy in living cells.
In macroautophagy, a portion of cytoplasm is wrapped with a membrane called separation membrane at first, thereby forming a vesicle called autophagosome (having a diameter of about 1 μm). Then, the autophagosome is fused to a lysosome, whereby the incorporated cytoplasmic materials are then degraded. Among autophagy-related proteins that have heretofore been found, a protein related to autophagosome formation and localized in the membrane, such as LC3, is known. Thus, such protein is fused to a fluorescent protein and expressed in cells, and autophagy is mesured by monitoring the accumulation of the fusion protein in the vesicular structure or the decrease in fluorescence intensity caused by degradation in the lysosome (Mizushima, N., Int. J. Biochem. Cell Biol., Vol. 36, pp. 2491-2502, 2004; and Shvets, E. et al., Autophagy, Vol. 4, pp. 621-628, 2008).
However, because the formation of autophagosome is a phenominon observed only in the case of macroautophagy, it is impossible to detect microautophagy or chaperone-mediated autophagy by the method as mentioned above. In the case of microautophagy or chaperone-mediated autophagy, vesicles for transfer, such as autophagosome, are not formed, and cytoplasmic materials are thought to be directly incorporated into the lysosome. At present, however, research thereon has not advanced as that of macroautophagy, and there are no effective methods for measurement. It is thus impossible to determine the total amount of all types of autophagy occurring in cells.
While the pH in the cytoplasm is neutral (pH, around 7), the pH in the lysosome or vacuole in which cytoplasmic materials are degraded by autophagy is acidic (pH, around 4). There is a method that, through utilizing such pH properties, autophagy can be detected based on pH-dependent changes in fluorescent properties caused by transfer of a fluorescent probe reagent resistant to degrading enzymes to the lysosome or vacuole. Because the cytoplasmic materials are ultimately incorporated into the lysosome or vacuole in all types of autophagy, the total amount of autophagy can be measured by this method. For example, Rosado et al (Rosado, C. J. et al., Autophagy, Vol. 4, pp. 205-213, 2008) use a probe reagent prepared by ligating, via a linker peptide, a fluorescent protein (DsRed.T3) that emits fluorescence at a relatively constant level independent of pH changes in the environment, to a fluorescent protein (super ecliptic pHluorin) that exhibits lowered fluorescence intensity as pH becomes more acidic. DsRed.T3 is a fluorescent protein that emits red fluorescence (587 nm), and the super ecliptic pHluorin is a fluorescent protein that emits green fluorescence (508 nm). Such probe is expressed in the cytoplasm, pH changes that occur when the probe is incorporated into the lysosome together with other cytoplasmic materials are measured as changes in a ratio of intensities of two fluorescences having different colors, thereby determining the activity of autophagy.
Because Rosado et al employ two fluorescent proteins, there is a problem that their technique makes the accurate measurement of autophagy activities difficult for the following reasons: that because the size of a label becomes large, the activity or localization of the proteins may probably be inhibited due to steric hindrance when the fluorescent protein is fused to a target protein; that two fluorescent proteins may probably generate improper signals when they are cleaved with protease in cells; that because pH-independent fluorescence properties of the two fluorescent proteins differ from each other in terms of quenching properties caused by the folding speed or photobleaching in cells, the value of the ratio may probably vary depending on experimental conditions; that because changes in the ratio depend only on fluorescence changes of the super ecliptic pHluorin, significant changes cannot be observed; and the like.